PROJECT SUMMARY There are many factors that govern the potential for arbovirus emergence and expansion, including host factors and regional environmental conditions, yet the foremost drivers of emergence and spread are the interactions between virus and vector. Defining these interactions requires in vivo experimental studies in natural hosts. Zika virus (ZIKV; Flaviviridae, Flavivirus) is the latest in a series of successful arbovirus invasions in the Americas. The primary goal of the proposed studies is to utilize experimental evolution and infection in relevant field populations of U.S. mosquitoes to identify the emergent and adaptive potential of ZIKV in the U.S. The secondary goal is to gain a broader understanding of selective pressures and vector- virus interactions between ZIKV and mosquitoes and to commence mechanistic studies to interrogate the mechanistic basis of ZIKV transmission. The adaptive potential of RNA viruses is well established, and there are documented examples of adaptive evolution in mosquitoes facilitating epidemiologically significant shifts with many arboviruses, including DENV, WNV, and CHIKV. Unlike these well-studied pathogens, much of the basic biology of ZIKV is not well established, making predicting how genetic change might alter transmission patterns and intensity more difficult. This study is designed to comprehensively test the hypothesis that significant constraints on ZIKV adaptation and emergence in the U.S. are likely to limit high levels of local transmission in the short term, yet also that the potential for the evolution of more adaptive strains is high and increasing with increased frequency of introduction and expanding populations of competent vectors.